广西红树林土壤放线菌新型抗肿瘤活性物质研究

Actinomycetes are an important source of anticancer drugs , so far , microbial secondary metabolites have been found in the physiological activity of 50% from ordinary soil actinomycetes , and special habitats actinomycetes in the environment to adapt to the long-term evolutionary process , it formed a unique metabolic pathways and secondary metabolites in different terrestrial actinomycetes , which become a source of novel anti- cancer compounds. Mangroves as a special marine environment , is not yet well developed , and a class of microbial repository and the use of new compound libraries . This study was preliminary and obtained more than 600 strains of actinomycetes freshly isolated from soil of mangroves in Guangxi found objects , using the rice blast fungus (P.oryzae) high-throughput screening model for screening , rescreening employing cancer cells in vitro , determine the antitumor activity of strains , has been screened out more than 10 strains of currently active strains , and its classification and identification . After positive strains carry out optimization of fermentation conditions , chromatographic separation of secondary metabolites of anti-tumor activity of the compounds were obtained in a two-dimensional structure of modern NMR and high resolution mass spectroscopy analysis based compounds found strong activity of new natural antitumor compounds ; and carry out its anti-tumor mechanism of action studies to explore novel anti -tumor activity of substances with potential applications .

放线菌是抗肿瘤药物的重要来源，迄今为止，已发现的具有生理活性的微生物次级代谢产物有50%来自普通土壤放线菌,而特境放线菌在适应环境的长期进化过程中，形成的独特代谢途径使其次级代谢产物与陆生放线菌不同，从而成为新型抗肿瘤化合物的来源。红树林作为特殊的海洋生态环境，是一类尚未很好开发和利用的微生物资源库和新化合物库。本研究拟用前期从广西红树林土壤获得600多株以及新分离的放线菌为发现对象，采用稻瘟霉（P．oryzae）筛选模型进行高通量初筛，用人癌细胞体外复筛，确定抗肿瘤活性菌株，目前已筛出10多株具有活性的菌株，并对其进行分类鉴定。阳性菌株开展发酵条件优化后，进行抗肿瘤次级代谢产物的色谱分离，获得的活性化合物进行以二维核磁共振和高分辨质谱等现代波谱学为主的化合物的结构解析，发现强活性新型天然抗肿瘤化合物；并开展其抗肿瘤作用机制的研究，发掘具有潜在应用前景的新型抗肿瘤活性物质。

我们在广西北仑河口国家级保护区和茅尾海红树林区级保护区采集土壤样本34份，采用10种分离培养基，以稀释涂布法分离并纯化放线菌，通过PCR扩增和测序获得菌株16ＳrRNA基因序列，并在数据库中搜索比对，进行分子鉴定，从土壤共获得的559 株放线菌；并分析放线菌的多样性：放线菌分布在7个目9个科15个属，其中小单孢菌属（Micromonospora）和链霉菌属为优势菌属，占所分离放线菌总数的50% ；对放线菌进行发酵，分别取发酵液水相、酯相、菌丝浸提液３类样品；采用纸片扩散法（ＫＢ法）测定其抗菌活性；以铜绿假单胞菌（ATCC 27853）及耐药株（2774）、大肠埃希菌（ATCC 25922）及耐药株（2800）等14株病原菌筛选抗菌活性菌株，共获得抗菌活性放线菌256株；特别值得注意的是，筛选出对MRSA（耐甲氧西林金黄色葡萄球菌）有抑制活性的菌株18株，对铜绿假单胞菌耐药株有抑制活性的菌株3株。 PCR检测活性菌株的抗生素生物合成基因，I型聚酮合酶(PKS I) KS域基因序列；II型聚酮合酶(PKS II)KS域基因序列；非核糖体多肽合成酶(NRPS)A结构域基因序列；对放线菌的抗生素生物合成基因探测发现，同时具有PKS I、PKS II及NRPS生物合成基因，至少含有其中1种，总阳性率91.30%。同时对具有抗菌活性的菌株进行抗人癌细胞体外筛选试验，通过MTT来测定抗肿瘤活性IC50(µg/mL)，以HeLa、MCF-7、HepG2三种细胞来检测抗肿瘤活性，结果有22株放线菌具有抗肿瘤活性。获得9个潜在新种放线菌菌株，如N04M112、3804M13、Z05M23和Z07M34与其同属内最近的有效发表菌株的16S rRNA基因序列相似性都低于98.65%，分别为97.49%、98.42%、98.58%和98.54%，可能为潜在新物种，目前采用多项分类对其进行鉴定中。发掘具有潜在应用前景的抗肿瘤活性物质，为广西红树林放线菌资源开发提供线索和依据。参加国内学术会议2次，申请发明专利2项，获得新型实用专利2项，发表科研论文7篇。项目按计划顺利进行,已经完成各项指标。